Method for amplification-free nucleic acid detection on optofluidic chips

ABSTRACT

An optofluidic platform is constructed so as to comprise a planar, liquid-core integrated optical waveguides for specific detection of nucleic acids. Most preferably, the optical waveguides comprises antiresonant reflecting optical waveguide (ARROWs). A liquid solution can be prepared and introduced into the optofluidic platform for optical excitation. The resulting optical signal can be collected at the edges of the optofluidic platform and can be analyzed to determine the existence of a single and/or a specific nucleic acid.

CROSS REFERENCE

This application is a divisional of U.S. patent application Ser. No.13/988,217 filed May 17, 2013, which is a 371 application ofPCT/US2011/061484 filed Nov. 18, 2011, which claims benefit under 35U.S.C. §119(e) of U.S. Provisional Application 61/415,482 filed on Nov.19, 2010, the contents of which are hereby incorporated by reference intheir entirety.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No.R01-EB006097 awarded by the National Institutes of Health/NationalInstitute of Biomedical Imaging and Bioengineering. The government hascertain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to the field of integratedoptics, and more particularly to an optofluidic platform for opticalparticle detection without the need for advanced microscopy equipment.The optofluidic platform can comprise planar, liquid-core integratedoptical waveguides for specific detection of nucleic acids. The opticalwaveguides can employ antiresonant reflecting optical waveguides, knownas ARROWs or ARROW waveguides.

BACKGROUND

Nucleic acid testing (NAT) is an essential part of the rapidly growingfield of molecular diagnostics (MDx). It allows for patient specificdiagnostics on the genome level as well as for perfect identification ofpathogens, e.g. discrimination between different virus strains.

The current gold standard for nucleic acid testing of viruses and otherorganisms is real-time polymerase chain reaction (RT-PCR) followed bysequencing. RT-PCR requires highly skilled operators, expensive reagentsand tightly controlled reaction environments. This is largely due to theneed for amplification of viral nucleic acids to generate large enoughsignals for readout. These limitations suggest a critical need for a newtype of diagnostic instrument for amplification-free viral detectionthat is rapid, sensitive, reliable, and quantitative.

SUMMARY

We introduce a different approach to nucleic acid testing based onplanar optofluidics—the combination of both integrated optical andfluidic components in the same miniaturized system. This approach usesplanar, liquid-core integrated optical waveguides for specific detectionof nucleic acids. This novel strategy enables the construction ofcompact, planar devices with sufficient sensitivity to detectfluorescently labeled nucleic acids from small (microliters) samplevolumes without the need for costly and time-consuming targetamplification. The simultaneous emphasis on vertical functionalintegration of optical and fluidic capabilities permits interfacing thedetection element with standard fiber optics and microfluidics. Thecombination of these innovative aspects eliminates the key obstacles toversatile point-of-care viral analysis for a multitude of applicationsin clinical settings, biomedicine, analytical chemistry and otherfields.

In a presently preferred embodiment of the invention, an optofluidicchip is constructed so as to comprise a self-contained, planaroptofluidic platform for optical particle detection. In a furtherembodiment, the optofluidic platform can comprise hollow-coreantiresonant reflecting optical waveguides (ARROWs), solid-core ARROWs,and fluidic reservoirs. The configuration of the different components ofthe optofluidic platform can allow liquids to be introduced into thehollow-core ARROWs and sub-picoliter volumes thereof to be opticallyexcited for single particle detection.

In an embodiment, a liquid solution can be introduced into theoptofluidic platform and can be optically excited to generate signal.The generated signal can be collected using, for example, a photodiodeand can be analyzed. The analysis can comprise determining the existenceof a fluorescence signal generated by a fluorophore attached to anucleic acid, which can indicate the existence of a single nucleic acidparticle contained in the liquid solution. As an example of afluorophore, a molecular beacon specific to a particular nucleic acidcan be prepared and introduced into the optofluidic platform. As such,the generated signal can indicate the existence of the specific nucleicacid. In an embodiment, the collected signal can be further analyzedusing techniques such as a fluorescence correlation spectroscopy.

Other aspects of illustrative embodiments of the invention are describedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Planar optofluidic platform. (a) schematic layout, images ofwaveguide cross sections and completed chip; (b) key microfabricationprocess steps for creating liquid-core waveguides.

FIG. 2: Concepts of (a) molecular beacon, (b) fluorescence resonanceenergy transfer (FRET).

FIG. 3: On-chip virus detection, (a) Q-B phage capsid labeled with Alexadye; (b) fluorescence signal recorded with virus in ARROW channel.Bursts indicate single particle detection events (inset: bufferbaseline); (c) corresponding autocorrelation G(r) of fluorescencefluctuations showing sub-single particle sensitivity (G(O)>1) andexcellent agreement with theory (red line; solid line).

FIG. 4: On-chip amplification-free virus detection, (a) strain-specificsegment of HPV-18 genome(http://www.ncbi.nlm.nih.gov/nuccore/9626069?report=graph&log$=seqview);(b) hairpin beacon structure with fluorescent label (F, TYE665) andquencher (Q, Iowa Black), on 5′ and 3′ ends, respectively; (c)fluorescence signal (red; solid line) for on-chip beacon detection at 1OpM target concentration (inset: background); (d) fluorescence signalversus target concentration I number of molecules in excitation volume.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Optofluidics is a rapidly growing field that deals with the interplay ofoptics and fluids, typically liquids, at the microscale. Currently, themajor research trends include optical devices defined by fluids, opticalparticle manipulation in liquids, and optical particle detection andanalysis, especially in biology and biomedicine.

We have invented an optofluidic approach to amplification-free nucleicacid testing that is based on liquid-core optical waveguides thatmaximizes the interaction between light and sample analytes. Based oncreating hollow-core antiresonant reflecting optical waveguides(ARROWs), we have developed a self-contained, planar optofluidicplatform for optical particle detection with extremely high sensitivitybut without the need for advanced microscopy equipment. The basic layoutof this platform along with the fabrication steps for forming thehollow-core waveguides are shown in FIG. 1.

The scanning electron image in the bottom center of FIG. 1 shows a crosssection of such a waveguide with hollow-core dimensions of 5×12 μm. Inaddition, solid-core ARROW waveguides (see SEM in bottom right of FIG.1a ) are connected to different points of the liquid core. This createsseparate access paths for liquids and light into the main channel, andcan also be used to define optical excitation areas with sub-picolitervolumes to achieve single molecule sensitivity. FIG. 1(a) depicts atypical experimental layout in which excitation light (green; arrowpointing into the optofluidic platform) enters the liquid core throughan orthogonally intersecting solid-core ARROW. Generated light (red;arrow pointing out from the optofluidic platform) is collectedperpendicularly in the chip plane and guided to the chip edges fordetection. Fluidic reservoirs at the channel ends allow for easy channelfilling and insertion of electrodes to induce electrokinetic particlemovement. The photograph in the bottom left of FIG. 1(a) illustrates thecompact size of a completed optofluidic chip.

The fabrication process shown in FIG. 1(b) includes (i) deposition ofdielectric layers (e.g. SiO2 and SiN) of the correct thickness on asilicon substrate; (ii) patterning of a sacrificial material (e.g. SU-8)into the desired hollow-core shape; (iii) covering the sacrificial layerwith additional ARROW guiding layers; and (iv) removal of thesacrificial core with chemical etching after exposing its ends by plasmaetching. It can be used flexibly to define a variety of optical andfluidic layouts with microscale precision.

The platform depicted in FIGS. 1(a) and 1(b) has successfully been usedfor detection and analysis of a variety of molecules, includingfluorescence detection of single dye molecules, fluorescence correlationanalysis of liposomes and ribosomes, and surface-enhanced Ramandetection of rhodamine 6G molecules.

This invention disclosure introduces the use of the optofluidic platformfor amplification-free molecular diagnostics of viruses.

Such a method requires both a suitable optical readout mechanism andsufficient detection sensitivity.

Optical Virus Detection

Optical detection methods play a large role in viral detection. Amongthese, fluorescence-based techniques are dominant. Typically, dyemolecules or semiconductor quantum dots that efficiently re-emit lightat a longer wavelength after optical excitation are attached to thetarget substance. Two advanced fluorescence methods used for virusdetection, and of relevance to this application, are molecular beaconand FRET detection. FIG. 2a illustrates the principle of a molecularbeacon, a short sequence of oligonucleotides that is furnished with afluorescent dye and a quencher molecule at its ends. In the “off” state,the beacon forms a hairpin bringing fluorophore and quencher into closeproximity and preventing fluorescence. When binding to a matchingsequence on a DNA or RNA target, the beacon opens and hybridizes whichresults in fluorescence emission from the now unquenched dye. Beaconshave the advantage of low background signal (no fluorescence signal inhairpin conformation) and high sensitivity for single base-pairmismatches. A drawback is the potential to unfold, especially in thepresence of enzymes and proteins. Beacons are commercially availableonce suitable nucleotide sequences are provided.

The principle of fluorescence resonance energy transfer (FRET) appliedto the identification of genetic material is shown in FIG. 2(b). Two dyemolecules (donor D, acceptor A) are attached to short nucleotidesequences. Non-radiative energy transfer between donor and acceptor canlead to acceptor fluorescence even if only the donor is excited. Theefficiency of this energy transfer depends strongly on the proximity ofthe two dyes, and a measurable acceptor signal is only observed whenboth probes are bound to a target with matching sequence (bottom).

Both molecular beacons and FRET detection create a detectablefluorescence signal with high specificity. In addition, both techniqueshave successfully been used for single molecule analysis and forfluorescence-based virus detection.

Molecular beacons and FRET are two examples for how nucleic acidspecific optical signals can be created for detection on the optofluidicchip.

High-Sensitivity Detection On Integrated Chip

The second key requirement for amplification-free detection is theability to detect fluorescence of biological samples at the singleparticle level. Of particular interest in this context is our recentdemonstration of ultrasensitive virus detection. Fluorescently labeledQ-E bacteriophage viruses (FIG. 3(a)) in solution were introduced intoour optofluidic chip through fluid reservoirs at one end of thehollow-core channel and optically excited at the intersection betweenliquid- and solid-core waveguides. The generated signal was collectedalong the liquid core waveguide (see FIGS. 1(a) and 1(b)) and results inclear fluorescence bursts due to single virus particles (FIG. 3(b)) thatare not present if pure buffer solution is tested (inset to FIG. 3(b)).The single particle sensitivity is confirmed by the fluorescencecorrelation spectrum (FIG. 3(c)) where a signal above one indicates lessthan a single particle in the excitation volume. The correlation signalalso allowed us to extract the diffusion coefficient of the detectedparticles which showed good agreement with the reported value for Q-B,further corroborating the successful detection of viral particles.

To date, this is the only demonstration of single virus detection on achip without the use of a microscope, and establishes planar optofluidicdetection as a suitable method for highly sensitive bioparticledetection. However, a second necessary step is to demonstrate specificdetection of a virus type and strain. To this end, we designed amolecular beacon specific for the LI gene of human papillomavirusHPV-18. The relevant region within the HPV genome and the 30 mer beaconstructure are shown in FIGS. 4(a) and 4(b), respectively. Molecularbeacons were mixed with matching oligonucleotides of variousconcentrations. Beacon binding was facilitated by brief heating (95° C.)followed by a cool down period (50° C. for 3 min). Beacon fluorescencewas then excited with 2 mW of laser light at 633 nm and detected asshown in FIG. 1(a). The resulting signal is depicted in FIG. 4(c) andshows clear beacon fluorescence followed by photo bleaching whencompared to the target-free negative background (inset), even at thelowest concentration of 1 OpM. FIG. 4(d) displays the signal dependenceon target concentration and corresponding average number of molecules inthe excitation volume. It shows both a linear behavior and demonstratesunequivocally our capability to detect viral DNA using molecular beaconfluorescence on the planar optofluidic ARROW platform with singlemolecule sensitivity.

We note that while these results clearly show the ability to detectnucleic acids specifically in an optofluidic device, the data of FIG. 4were measured in a “static” configuration where the channel contentswere not moving. The preferred implementation for the amplification-freenucleic acid detector would be to detect nucleic acids within a movingstream of analyte liquid that is moving past the excitation point.

The generated signal described above can be collected using lightsensors, such as a photodetector. For example, a photodiode can be usedto capture and convert the fluorescence signal generated along theliquid-core waveguide to a current or a voltage used for analyzing thefluorescence bursts and for determining the existence of specificnucleic acids. As a further example, an avalanche photodiode can also beused to convert the generated light to electricity. Being asemiconductor highly sensitive to light, the avalanche photodiode can beconfigured to provide a highly accurate signal representation of thegenerated light.

We claim:
 1. A system for detecting single nucleic acid particles in aliquid solution, comprising: a planar optofluidic platform; a lightsource arranged to optically excite a liquid solution introduced intothe planar optofluidic platform, whereby a fluorescence signal isgenerated; and a light sensor arranged to detect the fluorescence signaland produce an electric signal for determining the presence of aspecific nucleic acid.
 2. The system of claim 1, further comprisingmeans for determining that the generated signal comprises fluorescencebursts.
 3. The system of claim 1, wherein the planar optofluidicplatform comprises planar, liquid-core integrated optical waveguides. 4.The system of claim 1, wherein the planar optofluidic platform compriseshollow-core antiresonant reflecting optical waveguides (ARROWs),solid-core ARROWs connected to different points of the hollow-coreARROWs, a first port for introducing liquid solutions in the hollow-coreARROWs, a second port for introducing excitation light to thehollow-core ARROWs, and a third port for collecting the generatedfluorescence signal.
 5. The system of claim 4, wherein the first portcomprises a fluidic reservoir contained in the planar optofluidicplatform.
 6. The system of claim 4, wherein the third port is configuredfor collecting at edges of the planar optofluidic platform generatedlight perpendicular to the planar optofluidic platform.
 7. The system ofclaim 1, wherein the light sensor comprises a photodetector or avalanchephotodiode.
 8. An optofluidic system for amplification-free detection ofa specific nucleic acid sequence in a liquid solution, comprising: aplanar hollow-core antiresonant reflecting waveguide (ARROW); a planarsolid-core ARROW intersecting the hollow-core ARROW at a sub-picoliteroptical excitation region; first and second fluidic reservoirs at endsof the hollow-core ARROW configured for introducing a liquid solutioninto the hollow-core ARROW, the liquid solution comprising a specificnucleic acid sequence and a nucleic acid probe labeled withfluorophores, wherein the nucleic acid probe specifically hybridizeswith the specific nucleic acid sequence in the liquid solution; a lightsource configured for providing an excitation light to the opticalexcitation region; and a light sensor configured for collecting afluorescent signal generated in the optical excitation region, whereinthe fluorescent signal is indicative of the presence of the specificnucleic acid sequence.
 9. The system of claim 8, further comprisingmeans for inducing electrokinetic particle movement to the liquidsolution in the one of the hollow-core ARROWs.
 10. The system of claim8, wherein the solid-core ARROW is configured to provide a path for theexcitation light to enter the hollow-core ARROW at the opticalexcitation region, and a path for collecting a fluorescent signalperpendicularly to the plane of the planar optofluidic system and forguiding the fluorescent signal to edges of the planar optofluidic systemfor detection.
 11. The system of claim 8, further comprising a secondhollow-core ARROW, a port for introducing a pure buffer solution free ofnucleic acids into the second hollow-core ARROW, and second solid-coreARROW intersecting the second hollow-core ARROW, wherein a fluorescentsignal generated from the liquid solution in the optical excitationregion can be compared to a signal from the pure buffer solution. 12.The system of claim 8, wherein the light source comprises a laser. 13.The system of claim 8, wherein the light sensor comprises aphotodetector or avalanche photodiode.
 14. The system of claim 8,further comprising means for creating a fluorescence correlationspectrum using the fluorescent signal.
 15. The system of claim 8,further comprising means for creating a plot of the fluorescent signal.